Process for the synthesis of cationic surfactants comprising esterification with basic character amino acids

ABSTRACT

The process of the invention is directed to the synthesis of cationic type surfactant compounds consisting of natural basic-character amino acids, and any of their homologs, suitably modified for the purpose of obtaining products having specific applications as antimicrobial (biocidal) agents. The process comprises a first step of esterification of an amino acid, and a second step of the condensation of a fatty acid chloride with an esterified amino acid derivative. Nontoxic reaction media and catalysts are used, and a final product free from impurities is obtained. The cost of the process of the invention is reduced from prior art processes due to the use of cheaper starting materials and simpler equipment.

This application is a 371 of PCT/E595/00027 filed Mar. 8, 1995.

The present invention relates to a new process for obtaining cationicsurfactant products, the hydrophilic portion of which consists of anesterified basic character amino acid and the hydrophobic portionthereof consists of a fatty acid linked to the α-amino group of theamino acid via an amide bond.

BACKGROUND OF THE INVENTION

The application of cationic surfactant compounds as antimicrobial agentshas been studied extensively.

As background to the present patent application, the following patentsmay be mentioned: JP 7416005; JP 7505350; JP 723571; JP 7783942; JP73118516; JP 8153280; GB 1352420, and U.S. Pat. No. 3,985,722.

There is a history of application of compounds of a similar nature infields such as cosmetics (see Patent GB 1352420), dentrifices (JP51023571) and hair conditioners (GB 2140297).

In Patents EP 320,976 and GB 1352420, the synthesis is described ofcompounds similar to the ones obtained in the process of the invention(such as pyrrolidinecarboxylate salts ). The synthesis process iscarried out in a particular order and according to particular reactionconditions: condensation of the fatty acid with the amino acid in anorganic/aqueous medium, subsequent esterification of the N-acylaminoacid with the corresponding alcohol saturated with hydrochloric acid andlastly salt formation with pyrrolidinecarboxylate.

In U.S. Pat. No. 3,985,722, the acylating reactant is a mixture of thefatty acid and sulfur trioxide, the reaction being carried out in thepresence of triethanolamine.

In Patent ES 512643, the preparation of the compounds which are thesubject of the present application comprises a first step ofesterification and a second step of condensation of an esterified aminoacid derivative with a fatty acid, the fatty acid being used directlywithout derivatization and dicyclohexylcarbodiimide being employed ascondensing agent.

SUMMARY OF THE INVENTION

The present invention involved a process for the synthesis of cationicsurfactants derived from the condensation of fatty acids with esterifieddibasic amino acids, of the formula: ##STR1## where: X⁻ : can be: Br⁻,Cl⁻, HSO₄ ⁻ sic!

R₁ : is a linear alkyl chain of a saturated fatty acid or hydroxy acidhaving 8 to 14 carbon atoms, linked to the α-amino group of the aminoacid via an amide bond.

R₂ : can be a linear or branched alkyl residue having 1 to 12 carbonatoms or an aromatic residue.

R₃ : can be ##STR2## it being possible for n to vary from 0 to 4, wherethe process comprises a first step of esterification of an amino acid,characterized in that, in a second step, condensation of a fatty acidchloride takes place with an esterified amino acid derivative.

The process to which this patent relates consists of at least two steps,which are as follows:

1.--Esterification of the carboxyl group of the α-amino acid withlinear, branched or aromatic C₁ -C₁₂ alcohols, using thionyl chloride asreagent and exploiting the heat of reaction in order to carry it out.

2.--Condensation of the C₈ -C₁₄ fatty acid linear alkyl chain with theα-amino group starting from the corresponding acid chloride in analkaline aqueous medium.

The present invention utilizes nontoxic starting materials and catalystsand provides a cationic surfactant which is free from impurities.

The present invention encompasses a method of using the cationicsurfactant obtained according to the process described as anantimicrobial agent.

DESCRIPTION OF THE INVENTION

The objective of the present invention is to prepare cationicsurfactants derived from basic character amino acids in good yield andstate of purity from nontoxic starting materials and catalysts, the costin energy terms being minimized and a final product free from impuritiesbeing obtained, with specific applications as antimicrobial agents.

The present invention relates to the preparation of molecules whichcorrespond to the formula: ##STR3## where: X⁻ can be Br⁻, Cl⁻, HSO₄ ⁻

R₁ is a linear alkyl chain of a saturated fatty acid or hydroxy acidhaving 8 to 14 carbon atoms, linked to the α-amino group of the aminoacid via an amide bond.

R₂ can be a linear or branched alkyl residue having 1 to 12 carbon atomsor an aromatic residue.

R₃ can be ##STR4## it being possible for n to vary from 0 to 4.

The starting materials used can be:

technical grade amino acids.

technical grade fatty acid chlorides.

deionized water.

technical grade caustic soda and hydrochloric acid.

The process to which this patent relates consists basically of twosteps.

1.--Esterification of the carboxyl group of the α-amino acid withlinear, branched or aromatic C₁ -C₁₂ alcohols, using thionyl chloride asreagent and exploiting the heat of reaction in order to carry it out.

2.--Condensation of the C₈ -C₁₄ fatty acid linear alkyl chain with theα-amino group starting from the corresponding acid chloride in analkaline aqueous medium.

In the step of esterification of the carboxylic acid of an α-amino acid,any commercial amino acid may be used, preference being given to dibasicamino acids, especially L(+)-arginine.

The second step of condensation of the fatty acid chloride or hydroxyacid chloride with the amino acid ester is performed in an alkalineaqueous medium without the need to use any organic solvent. Saidcondensation is carried out at an alkaline pH, preferably at a pH ofbetween 8 and 10.

The alkaline pH is obtained by adding an alkali metal hydroxide,preferably sodium or potassium hydroxide, to the aqueous medium.

When the condensation has been performed, the product is recovered bymeans of centrifugation of the precipitated product after adjusting themedium to a practically neutral pH, preferably to a pH of between 6 and7, by adding an inorganic acid, preferably hydrochloric acid.

Preferably, in the process of the invention, the esterification of theamino acid with C1-C12 alcohols, especially ethanol, takes place byadding thionyl chloride to a suspension, prepared at room temperature,of arginine in alcohol.

The process of the invention differs from the previous processes both inthe esterification step and in the condensation step.

In the esterification step, the same reactants and the same catalyst areused as in Patent ES 512643, but the reaction sequence is different. Thereactants (arginine and ethanol) are not mixed simultaneously with thecatalyst (thionyl chloride), instead the catalyst is added subsequentlyto the reactants. In this way, the reaction of the invention isexothermic, so that the heat evolved is exploited with a correspondingenergy saving during this phase of the process.

In the condensation step, the components are different. Thus, forexample, in Patent ES 512643, DMF (dimethylformamide),dicyclohexylcarbodimide and a fatty acid are used, while, in theinvention, water, caustic soda and a fatty acid chloride are used. Thisstep thus entails a condensation of the fatty acid chloride in anaqueous medium, the hydrochloride of the corresponding derivative beingseparated.

Consequently the process of the invention differs substantially from theprevious processes as regards the nontoxic starting materials andcatalysts used and the freedom from impurities in the final productsobtained, a very important discriminatory feature inasmuch as iteliminates the interference which said impurities may produce in thefinal application as antimicrobial agents. Due to the materials used,the cost of the process also proves lower and the equipment simpler.

The present invention also relates to the application of the productsobtained by the above-mentioned process as antimicrobial (biocidal)agents.

The products described lack skin irritant effects and significantgastric ulcerogenic activity, are not mutagenic (according to the Amestest) at the doses at which they are normally used in their fields ofapplication and possess LD₅₀ values via the oral route of greater than2000 mg/kg. (The LD₅₀ is a way of expressing the toxicity of anyproduct, and is defined as the minimum dose, expressed in mg of productunder study per kg of test animal bodyweight, which produces the deathof 50% of the animals which are the subject of the test).

The products described are capable of forming supramolecular aggregatesof the micelle type, liquid crystals, emulsions and microemulsions inbinary, ternary and quaternary systems, the technology of which isapplicable to many industrial fields such as cosmetics, dermopharmacy orfoodstuffs.

EXAMPLES

Several examples are detailed below:

One example of obtaining a product and four examples of application: twoin the meat industry and two in cosmetics.

Example I

We shall describe the process for obtaining, on a laboratory scale, aspecific product: the lauramide of L(+)-arginine ethyl estermonohydrochloride.

As mentioned in the description, the process consists of two steps.

FIRST STEP

Preparation of L(+)-arginine ethyl ester dihydrochloride

In a glass reactor of capacity 2 liters with a five-socket lid andprovided with a mechanical stirrer, reflux condenser, nitrogen gasinlet, dropping funnel and thermometer, 1 equivalent of L(+)-argininehydrochloride is suspended in 200 ml of essentially water-free ethylalcohol at room temperature, and the stirrer is started.

1.3 equivalents of thionyl chloride are then added dropwise over aperiod of two hours, reflux being maintained by heating. When themixture reaches the boiling point, it is stirred for a further threehours, after which the solvent is removed by evaporating it off atreduced pressure several times, with prior additions of dry ethanol.

SECOND STEP

Preparation of the lauramide of L(+)-arginine ethyl estermonohydrochloride

The above crude reaction product is dissolved in water and neutralizedwith aqueous sodium hydroxide, and the mixture is subsequently broughtto pH 8-10 at which it is maintained for the remainder of the reactionwhile 1.1 equivalents of lauroyl chloride are added dropwise, thetemperature of the mixture being maintained below 20° C. by means of anappropriate cooling bath with ethylene glycol.

When the addition is complete, stirring is maintained for a further twohours, the pH being finally adjusted to values of 6-7 with hydrochloricacid. Lastly, the crude reaction product is filtered off, a white solidof pearly appearance being obtained, of yield 80-85% W/W with respect tothe product initially expected.

Example II

We shall describe the application of the product obtained according tothe process described in Example I as preservative in the meat industry,specifically of cooked ham.

With the object of evaluating the antimicrobial activity of the productobtained according to the process described in Example I, a test wasperformed on an industrial scale in a cooked ham production plant, asexplained below:

The product (the lauramide of L(+)-arginine ethyl estermonohydrochloride) is added into a 100-l reservoir containing injectionbrine for hams, in an amount such that it results in a dose of 2 g ofproduct per 1000 g of treated ham.

According to the usual industrial methods, the hams are injected withsaid brine, massaging is then performed for 48 hours using vacuum drumsand the hams are subsequently cooked (at 69° C. in the center of thepiece), after which they are wrapped. From this point on, amicrobiological study is carried out over four months, during whichperiod the pieces have been subjected to extreme storage conditions.

The evaluation of the efficacy of the product as preservative of hams(through its antimicrobial effect) has been carried out by means ofmicrobiological tests, the level of microbial (specifically bacterial,in this case) contamination present in the pieces being determined.

The method of determination is that of counting streaks on plates, totalmesophilic aerobic bacteria, enterobacteriaceae and heterolacticmicrobiota being determined.

The results obtained at the fourth month are as follows:

    ______________________________________                                                          CFU/g                                                                         N.P.   W. Prod.                                             ______________________________________                                        Total mesophilic aerobic                                                                            2 × 10.sup.5                                                                   1.5 × 10.sup.3                             Enterobadteriaceae  3.5 × 10.sup.3                                                                   absence                                          Heterolactic          1 × 10.sup.2                                                                   absence                                          ______________________________________                                    

(The results are expressed in colony forming units (CFU) (bacteria) pergram)

The column headed N.P. corresponds to product with no preservative; inthe column headed W.Prod. the product which is the subject of theexample has been added at the dose described.

From the results obtained, a clear picture emerges of the efficacy ofthe product, which significantly lowers the level of the firstmicroorganism mentioned (to levels which are tolerable for the ham notto putrify) and eliminates them completely in the other two cases.

Example III

This example consists of a repetition of the previous test, injectingthe hams with brine containing the product which is the subject ofExample II (W.Prod. column) and, by way of comparison, in another groupof hams, with brine containing a traditional chemical preservative basedon potassium sorbate and propyl p-hydroxybenzoate (TP column).

The results obtained are as follows:

    ______________________________________                                                          CFU/g                                                                         N.P.   W. Prod.                                             ______________________________________                                        Total mesophilic aerobic                                                                          1 × 10.sup.3                                                                     absence                                          Enterobacteriaceae  absence  absence                                          Heterolactic        absence  absence                                          ______________________________________                                    

from which there emerges a better efficacy of the proposed product thanthat of the traditional chemical preservative used as reference. This isin agreement with the good properties of the proposed product, given,moreover, its safety features.

It should be noted that the absolute value of the counts may vary fromone test to another, it being necessary to take into account the trends.

Example IV

We shall describe the application of the product in the cosmeticindustry, specifically its use as preservative in the preparation of ashampoo.

An amount (5 Kg) of shampoo is prepared on a laboratory scale accordingto a conventional formulation, expressed in % w/w:

    ______________________________________                                        25% Sodium lauryl sulfate                                                                              30%                                                  Lauric diethanolamide    5%                                                   Preservative (the same as in Ex. I and II)                                                             0.04-0.05%                                           Deionized water          q.s. 100%                                            ______________________________________                                    

(neither perfume nor colorant is included since these do not affect thepreservation process).

Another similar amount of shampoo is prepared with the same formula butwithout preservatives for use as reference.

The antimicrobial activity of both is determined by means of anadaptation of the "challenge" test, in which samples of both shampoosare arranged, adding to all of them a combined inoculum of yeasts,bacteria and fungi of the following species and strains:

Escherichia coli (ATCC No. 9027)

Staphylococcus aureus (ATCC No. 8739)

Pseudomonas aureoginosa sic! (ATCC No. 9077)

Candida albicans (ATCC No. 10231)

Aspergillus niger (ATCC No. 16404)

The test consists in contaminating a certain amount of the shampoo ofthe abovementioned samples by inoculating them with several knownmicroorganisms, and seeing their development at a particular time andtemperature (37° C. in our case).

In our case, the plate counts are performed at various incubation times,giving the results detailed below, from which the effectiveness of theproduct in the shampoo is clearly deduced (from the fall in the numberof colonies) by comparing the shampoo with the reference not containingthe product.

    __________________________________________________________________________              COLONIES /GRAM FORMULATION                                                    1st inoculation                                                     INCUBATION TIME                                                                         0 hours 24 hours                                                                              3 days  7 days                                      SAMPLE    TNB F & Y                                                                             TNB F & Y                                                                             TNB F & Y                                                                             TNB F & Y                                   __________________________________________________________________________    Reference 3 × 10.sup.6                                                                2 × 10.sup.5                                                                3 × 10.sup.6                                                                1 × 10.sup.4                                                                3 × 10.sup.6                                                                5 × 10.sup.2                                                                6 × 10.sup.6                                                                <10                                     (without                                                                      preservatives)                                                                With 0.4% of                                                                            3 × 10.sup.6                                                                2 × 10.sup.5                                                                <10 <10 <10 <10 <10 <10                                     the product                                                                   which is the                                                                  subject of the                                                                example                                                                       __________________________________________________________________________    where TNB = total number of bacteria                                          F & Y = number of fungi and yeasts                                                      COLONIES /GRAM FORMULATION                                                    1st inoculation                                                     INCUBATION TIME                                                                         0 hours 24 hours                                                                              3 days  7 days                                      SAMPLE    TNB F & Y                                                                             TNB F & Y                                                                             TNB F & Y                                                                             TNB F & Y                                   __________________________________________________________________________    Reference 5 × 10.sup.7                                                                3 × 10.sup.5                                                                5 × 10.sup.7                                                                5 × 10.sup.4                                                                7 × 10.sup.7                                                                4 × 10.sup.3                                                                7 × 10.sup.7                                                                1 × 10.sup.3                      (without                                                                      preservatives)                                                                With 0.4% of                                                                            5 × 10.sup.7                                                                3 × 10.sup.5                                                                4 × 10.sup.4                                                                1 × 10.sup.2                                                                <10 <10 <10 <10                                     the product                                                                   which is the                                                                  subject of the                                                                example                                                                       __________________________________________________________________________

Example V

In this example, a test is performed of its efficacy as preservative ina deodorant which is presented in aerosol form and whose formulation,expressed in % w/w, is as follows:

    ______________________________________                                        1,2-Propylene glycol       1%                                                 Ethanol                   40%                                                 Preservative (the same as in the previous ex.)                                                          0.4-0.5%                                            Deionized water           g.s. 100%                                           ______________________________________                                    

(perfume is not included since it does not affect the preservationprocess; neither is the propellant included)

The antimicrobial activity is determined by measuring the halo ofinhibition which are sic! produced in the agar plate cultures of thefollowing strains:

Pseudomonas aureoginosa (ATCC No. 9077)

Staphylococcus aureas (ATCC No. 8739)

Escherichia coli (ATCC No. 9027)

Candida albicans (ATCC No. 10231)

Aspergillus niger (ATCC No. 16404)

The plates are incubated in an incubator for 18 hours at 37° C.

The following results are obtained

    ______________________________________                                                     DIAMETER OF THE HALO                                                          OF INHIBITION                                                                 (+13 mm sample disk)                                                            Sample with 0.4%                                                              of the       Control sample                                    MICROORGANISM  preservative un-                                                                           (without                                          UNDER STUDY    der study    preservative)                                     ______________________________________                                        Staphyloccccus aureus                                                                        30           13                                                Pseudomonas aureoginosa                                                                      28           13                                                Escherichia coli                                                                             26           13                                                Candida albicans                                                                             15           13                                                Aspergillus niger                                                                            20           13                                                ______________________________________                                    

The results show the efficacy clearly: in the control samples a halo ofinhibition does not occur (13 mm does not count since this correspondsto the central sample-carrier disk), while those containingpreservatives produce a substantial-sized halo.

We claim:
 1. A process for the synthesis of cationic surfactants derivedfrom the condensation of fatty acids with esterified basic characteramino acids, of formula: ##STR5## wherein: X⁻ is selected from the groupconsisting of Br⁻, Cl⁻, or HSO₄ ⁻ ;R₁ is a linear alkyl chain of asaturated fatty acid or hydroxy acid having from 8 to 14 carbon atoms,wherein said alkyl chain is linked to the α-amino group of the aminoacid via an amide bond. R₂ is a linear or branched alkyl radical havingfrom 1 to 12 carbon atoms or is an aromatic radical; and R₃ is selectedfrom the group consisting of: ##STR6## wherein n varies from 0 to 4,said process comprising a first step of esterification of an amino acidwith C₁ -C₁₂ alcohols, comprising adding thionyl chloride to asuspension of arginine in alcohol at room temperature to produce anesterified basic character amino acid derivative, said process furthercomprising a second step, condensing a fatty acid chloride with theamino group of said esterified basic character amino acid derivative. 2.Process according to claim 1, characterized in that said basic characteramino acid derivative is L(+)-arginine.
 3. Process according to claim 1,characterized in that the second step of condensation of said fatty acidchloride with the amino group of the esterified basic character aminoacid derivative comprises using the chloride of said fatty acid, saidsaturated fatty acid having 8 to 14 carbon atoms.
 4. The processaccording to claim 1, in which the condensation of the fatty acidchloride or hydroxy acid chloride with the amino acid ester takes placein an alkaline aqueous medium.
 5. The process according to claim 3, inwhich the condensation of the fatty acid chloride or hydroxy acidchloride with the amino acid ester takes place in an alkaline aqueousmedium.
 6. The process according to claim 1, wherein the reaction mediumis maintained at a pH of between 8 and 10 by adding sodium hydroxide. 7.The process according to claim 4, wherein the reaction medium ismaintained at a pH of between 8 and 10 by adding sodium hydroxide. 8.The process according to claim 5, wherein the reaction medium ismaintained at a pH of between 8 and 10 by adding sodium hydroxide. 9.The process according to claim 1, further comprising adjusting the pH ofthe product to between 6 and 7 by adding hydrochloride acid to obtain acrude reaction product, and centrifuging said crude reaction product toseparate said cationic surfactant.
 10. The process according to claim 2,further comprising adjusting the pH of the product to between 6 and 7 byadding hydrochloride acid to obtain a crude reaction product, andcentrifuging said crude reaction product to separate said cationicsurfactant.
 11. The process according to claim 5, further comprisingadjusting the pH of the product to between 6 and 7 by addinghydrochloride acid to obtain a crude reaction product, and centrifugingsaid crude reaction product to separate said cationic surfactant. 12.The process according to claim 3, further comprising adjusting the pH ofthe product to between 6 and 7 by adding hydrochloride acid to obtain acrude reaction product, and centrifuging said crude reaction product toseparate said cationic surfactant.
 13. The process according to claim 4,further comprising adjusting the pH of the product to between 6 and 7 byadding hydrochloride acid to obtain a crude reaction product, andcentrifuging said crude reaction product to separate said cationicsurfactant.
 14. The process according to claim 6, further comprisingadjusting the pH of the product to between 6 and 7 by addinghydrochloride acid to obtain a crude reaction product, and centrifugingsaid crude reaction product to separate said cationic surfactant.
 15. Aprocess according to claim 1 wherein said esterified basic characteramino acid derivative is arginine ethyl ester dihydrochloride.